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Generation and characterization of anti-CD138 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Generation and characterization of anti-CD138 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
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Content
Generation and Characterization of anti-CD138 Chimeric
Antigen Receptor T (CAR-T) Cells for the Treatment of
Hematologic Malignancies
By
Nell Namitha Narasappa
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)
August 2016
Copyright 2016 Nell Namitha Narasappa
DEDICATION
To my grandfather, the late Dr. B.P Narasappa, for planting the seed of
scientific curiosity in my mind,
To my family, Narendra Narasappa, Vindhya Narendra, Naomi Niveditha,
and Jonathan Deepak,
For their unconditional love and unwavering support….
ACKNOWLEDGEMENT
I would like to express my sincere gratitude to Dr. Preet M. Chaudhary, my mentor
and advisor, for his constant support, encouragement and guidance during my research
project. Thank you for giving me this opportunity to be a part of your lab and work
towards the goals you have envisioned for the lab.
I would like to thank all the members of Dr. Chaudhary’s Lab for their generous
guidance and support during my research project. I would like to thank Dr. Sunju Choi
for guiding me through the molecular cloning procedures and Dr. Venkatesh Natarajan for
careful reading and providing suggestions to improve my thesis. I’m hugely indebted to
Dr. Ramakrishnan Gopalakrishnan and Dr. Hittu Matta for being excellent mentors and
guides throughout this endeavor.
I would like to thank Dr. Zoltan Tokes and Dr. Robert Maxson, members of my
thesis committee, for their support and encouragement. I would also like to thank Dr.
Zoltan Tokes for his insight and guidance throughout this two-year Master’s program, as
well as my academic advisor Jim Lee for his generous support and guidance.
I would like to thank God Almighty, my family and friends for standing by my side
through the ups and downs of graduate life and for their motivation and encouragement.
ABSTRACT
Chimeric antigen receptor (CAR) immunotherapy is a breakthrough in cancer therapy
because it allows for the in vitro re-engineering of specific T cell receptors against
different types of cancer. With CARs, glycolipid and carbohydrate tumor antigens can be
made recognizable by T-cells. Tumor cells become “visible” because of direct
recognition by T-cell receptor and moreover CAR is a targeting mechanism that functions
independent of MHC type recognition. A vast number of CARs have been developed
against hematological and non-hematological malignancies, but the success rate is higher
in hematological cancers. Among hematological malignancies, CAR T cells are already
in clinical trials to treat B-cell malignancies. Multiple myeloma (MM) is the second most
common blood cancer involving malignancy caused by cancerous plasma cells. Plasma
cell myeloma (PCM) is a type of multiple myeloma where abnormal plasma cells build
up in the bone marrow and lead to tumor formation in various bones of the body. These
cancerous cells crowd out the normal plasma cells that help fight infections leading to
impairment of immune function and kidney damage. The SEER (Surveillance,
Epidemiology, and End Results) data for multiple myeloma published by the National
Cancer Institute shows the average life expectancy at 4 years. Treatment for multiple
myeloma is focused on therapies that could decrease the clonal plasma cell population
and thus decrease the signs and symptoms of disease. There is a need for more effective
therapeutic options for MM. CD138 is a cell surface protein expressed specifically on
MM and plasma cells. An antibody targeted against this antigen can be successfully used
to engineer CAR-T cells for MM immunotherapy.
My hypothesis is that CD138 targeting CAR-T cells may have potent anti-MM activity.
1
Table of Contents
Introduction…………………………………………………………………………………..1
Structural Design of CARs……………………………………………………………3
Generation of CARs……………………………………………………………...........5
Components of successful immunotherapy of CARs…………………………………6
Mechanism of gene therapy with lentivirus vector…………........................................8
Other immune cells used in CAR………………………………………………........10
Natural killer cells……………………………………………………………….10
Jurkat-NFAT-eGFP T-cells…………………………………………………......10
Lymphoma…………………………………………………………………………...11
Multiple Myeloma……………………………………………………………….11
CD138 (Syndecan-1)……………………………………………………………13
Limitation of CARs………………………………………………………………….14
Objectives of Project..............................................................................................................16
Materials and methods……………………………………………………………..............17
Generation of lentiviral anti CD138-CAR construct………………..........................17
Virus generation…………………………………………………….........................18
Viral Transduction…………………………………………………………………..18
In vitro binding assay……………………………………………………………….20
Flow cytometry of Myc expression…………………………………………………20
Flow cytometry of GFP expression…………………………………………………20
In vitro cytotoxicity assay……………………………………………….………….21
ELISA……………………………………………………………………………….21
Results……………………………………………………………………………………….22
Construction of anti-CD138 CAR plasmids………………………………………...22
Binding Assay for detection of CD138 antigen on MM cell lines………………….24
Detection of Myc expression in CAR infected Jurkat cells………………………...26
Detection of GFP expression in CAR infected Jurkat cells………………………...28
Cytotoxicity Assay………………………………………………………………….31
Interleukin-2 Enzyme-linked Immunosorbent Assay (ELISA)………...…………...34
Discussion…………………………………………………………………………………...36
References…………………………………………………………………………………...38
List of Figures
Figure 1. The structure of CAR molecules…………………………………………….……..4
Figure 2. Generations of CAR molecules……………………………………………….……6
Figure 3. Components of successful immunotherapy of CARs………………………….…...8
Figure 4. Production of recombinant lentiviral vectors……………………………….……...9
Figure 5. One example of Jurkat cell activation………………………...…………………..11
Figure 6a. Production of abnormal plasma cells by the bone marrow leading to MM……....12
Figure 6b. Summary of CD138 reactivity in normal bone and bone related tumors………...13
Figure 7. Various limitations of CARs…………………………………………………...…..15
Figure 8a. Schematic representation of the CD138-CAR construct: pLenti-EF1∆Xho
CD138-MYC-hCD8-h4-1BB-CD3Ζ-T2A-Pac ………………………….…...….23
Figure 8b. Positive clones detected by colony-PCR……..………………………..……...….23
Figure 9. Binding assay to detect the cell line that possess maximum interaction with
CD138 scFv (antibody)…..…………….…………………………………….…..25
Figure 10. Flow cytometric analysis to confirm CAR expression on cell surface of
Jurkat-NFAT-eGFP cells…….……………………………………………….......27
Figure 11. Flow cytometric analysis of GFP expression in CAR infected Jurkat cells…..….30
Figure 12. Cell death assay of CAR-NK cells treated with cancer cells……………..……...32
Figure 13. Cell death assay of CAR-T cells treated with MM cancer cells……………..…...33
Figure 14. IL-2 cytokine secretion by Jurkat-NFAT-eGFP expressing CD138-CAR
cells upon activation with L363 cells………………………………………….....35
List of Abbreviations
CAR – Chimeric Antigen Receptor
MM – Multiple Myeloma
PCM – Plasma Cell Myeloma
SEER – Surveillance, Epidemiology and End Results
MHC – Major Histocompatibility Complex
APC – Antigen Presenting Cell
TAA – Tumor Associated Antigen
scFv – Single Chain Variable Fragment
Fc – (Fragment, crystallizable) region
[FCR]-γ chains – Fc gamma receptor
VSV-G – Vesicular Stomatitis Virus
CRS – Cytokine Release Syndrome
IL – Interleukin
PEL – Primary Effusion Lymphoma
XVIVO – Chemically defined, serum free, hematopoietic cell medium
NK – Natural Killer
CD138 – Cluster of Differentiation 138
HRP – Horseradish peroxidase
HEL – Human Erythroleukemia Cell Line
FACS – Fluorescence Activated Cell Sorting
eGFP – Enhanced Green Fluorescent Protein
Introduction
The immune system is the body’s defense mechanism that is entrusted with protecting the
host from harmful pathogens. The immune system is further classified as Innate Immune
system and Adaptive Immune system. The former is also known as non-specific/in- born
immunity and is responsible for responding and acutely reacting to pathogens. It provides
an immediate defense mechanism through barriers such as the skin, mucous membranes
and chemicals in the blood. The adaptive immune system, on the other hand is activated
by the innate immune system through the process of antigen presentation. This stimulates
the production of a specific class of white blood cells called the T-cells and B-cells.
These cells confer a long-lasting/protective immunity to the host wherein if a similar
attack were to take place again, the immune system will be activated much faster through
stimulation of the memory T-cells and B-cells.
Humoral immunity is conferred by B-cells that mature in the bone marrow and fight off
disease by the production of antibodies. Cell mediated immunity is conferred by T-cells
that mature in the thymus, and involves the activation of phagocytes, cytotoxic T-cells
and various cytokines that are produced in response to an antigen. T- cells possess T-cell
receptors that interact with the fragment of antigen bound to the major histocompatibility
complex (MHC) that are presented by antigen presenting cells (APCs). T-cells can also
recognize tumor-associated antigens (TAA) present in abundance on the tumor cells.
Upon recognition, activation of downstream signaling mechanisms occur leading to
apoptosis of the tumor cells.
Although these tumor-specific T-cells are effective in killing of tumor cells, they are rare
2
and require enrichment in order to effectively eliminate all tumor cells leaving no residual
tumor cells to prevent recurrence. A procedure called “Adoptive cell transfer (ACT)”
involves the isolation of this rare tumor reactive T-cells from patients, expansion ex-vivo
and then infusion into the cancer patient. It was widely sought after approach but came
with various technical limitations. This led to the development of Chimeric Antigen
Receptor (CAR) Immunotherapy.
CAR-T therapy involves genetic reprogramming of T-cells with artificial receptors
having specificity to known tumor antigens present on the malignant cells, enabling their
destruction by activating downstream T-cell effector mechanisms (Lipowska-Bhalla et
al., 2012). They assign monoclonal antibody specificity onto an immune effector cell and
this transfer occurs through the use of viral vectors carrying the specific coding sequence.
The tumor escape mechanism involves the down-regulation or modulation of MHC on
the tumor cell making it invisible to T cells (Garrido et al., 1993), as interaction between
T-cell receptor and MHC is essential for destroying the tumor cells. CAR-T therapy
allows circumventing the tumor escape mechanism as it functions independently of the
HLA antigen and antigen processing through the use of direct recognition of the antigens
present in abundance on the tumor cells. This therapy also circumvents the TCR
approaches where specific receptors have to be produced depending on the specific MHC
that the patient expresses. Also, CARs can be used on a range of potential protein targets
that include carbohydrate (Mezzanzanica et al., 1998) and glycolipid tumor antigens
(Kershaw et al., 2005, Murphy et al., 2005).
3
Structural design of CARs
CARs consisting of the antigen binding regions of a specific monoclonal antibody and
the α and β chains of TCRs were first developed in the mid-1980s (Kuwana et al., 1987).
Later in 1993, Zelig Esshar modified this design to incorporate an ectodomain; a single
chain variable fragment (scFv) from the antigen binding regions of both heavy and light
chains of a monoclonal antibody, a transmembrane domain and an endodomain
containing a signaling domain from CD3-ζ (Eshhar et al., 1993). The CARs developed
today still follow the same structural template.
The first step in CAR T-cell therapy is choosing an optimal TAA. The TAA should be
highly expressed on the surface of all tumor cells but lowly expressed/ not at all present
on important normal tissues (Newick et al., 2016). Depending on the TAA the CAR
construct is designed and generated. The CAR construct is composed of:
Ectodomain: refers to the portion of the receptor protruding from the surface of the T-
cell. It is composed of:
• Signal peptide: this peptide helps direct the nascent protein into the ER, assists in
glycosylating and anchoring the engineered receptor to the cell membrane.
• Antigen recognition region: it is usually a single chain variable fragment (scFv).
scFv region is derived from vL and vH region of an antibody bound by a linker,
and it binds the target antigen. Any component that binds the given target (tumor
associated antigen) with high affinity can be used in this region.
Hinge/Spacer: links the antigen-binding domain to the transmembrane domain, allows
for varying orientations that facilitate antigen recognition. Ex: hinge region from IgG1.
Transmembrane domain: a hydrophobic alpha helix that spans the membrane, different
4
domains result in different receptor stability.
Endodomain: responsible for transmission of the signal after the antigen is bound. CD3-
ζ is the most commonly used component and sometimes additional co-stimulatory
signaling is needed (Fig. 1). Upon interaction of the CAR with the antigen on tumor
surface, the endodomain/signaling domains facilitate the T-cell effector functions
including cytokines expression, secretion and lysis of the target cells.
Fig. 1. The structure of CAR molecules (Eshhar et al., 1993)
Single chain variable fragment (scFv) confers antigen specificity to a CAR and can be
derived from mouse monoclonal, humanized or fully human antibodies (Geldres et al.,
2016). Ideally a CAR should be designed to maximize interaction with tumor through the
TAA while minimizing toxicity to normal tissues. The hinge region provides flexibility in
5
interaction between the two while the transmembrane (TM) domain assists in anchoring
the CAR to the tumor cell surface. The CD28 TM portion is generally used along with
CD3-ζ as the activation domain. Co-stimulatory domains such as 4- 1BB are also used.
CD28 and 4-1BB co-stimulation can together, positively affect CAR persistence,
proliferation and effector functions.
Generations of CARs
First generation CARs are composed of an scFv derived from a monoclonal antibody that
is specific to the TAA. It is genetically engineered into a receptor comprising a TM and a
cytoplasmic activation domain (e.g., CD3-ζ or Fc receptor [FCR]-γ chains) (Park et al.,
2015). T- cell proliferation is very limited in first generation CARs and hence co-
stimulation was required for better survival and proliferation. Second generation of CARs
overcomes this by a modification in first generation CARs. T-cell costimulatory
molecules (e.g., CD28, OX40, 4-1BB) are incorporated in the CD3-ζ signaling domain,
this enhances T-cell proliferation and persistence. A clinical study using CD19-specific
CAR T-cells containing 4-1BB showed longer persistence and biological functions as
compared to first generation CARs was demonstrated (Mackall et al., 2014). Third-
generation CARs containing two co-stimulatory domains along with the CD3-ζ domain
are currently being tested (Mackall et al., 2014). CD28/OX40 or CD28/4-1BB are
examples of some co-stimulatory domains being used (Carpenito et al., 2009). As a result
of these additional domains, downstream kinase pathways that support gene transcription
and functional cellular response are activated (Geldres et al., 2016). Third generation
CARs have not demonstrated greater potency than second generation CARs and the
6
benefits are not very clear (Fig. 2). Fourth generation CARs are also being developed to
stimulate the innate immune system to assist in destroying the cancerous cells.
Fig 2. Generations of CAR molecules
The first-generation CARs contain an antigen-binding domain comprised of an scFv that
is linked to the CD3-ζ chain by a TM domain. Second- generation CARs incorporate a
co-stimulatory endodomain in addition to the CD3ζ domain. Clinical studies have shown
that co-stimulatory domains lead to enhanced expansion and persistence compared to
CARs that lack a co-stimulatory endodomain. Third-generation CARs contain two co-
stimulatory domains, in addition to the CD3ζ domain.
Components of successful immunotherapy of CARs
For successful CAR-T therapy, the CAR-T cells should be able to traffic to the tumor
site, interact and bind the specific TAA, proliferate and survival while stimulating T-
effector downstream signals to kill the target cells. These cells should also be able to
7
avoid inhibitory signals from the tumor microenvironment and facilitate
immunosurveillance of any residual tumor load. Before infusion of CAR-T cells into the
patient, lymphodepletion is advised using either chemotherapy or radiation. It is believed
to allow for homeostatic T-cell expansion and persistence by depleting cells that would
otherwise compete for available cytokines. CAR-T therapy tends to be more potent when
T-cell infusion occurs early after conditioning (Gill et al., 2014). T-cell dose is defined as
the total number of viable cells per body surface area/ per kilogram of ideal body weight,
and the optimal dose of total CAR positive cells remains unknown. Effective T-cell
trafficking depends on a variety of receptors, soluble factors and adhesion molecules
(Nolz et al., 2011), and it can be achieved by transduction/ electroporation to overexpress
the relative chemokine (Moon et al., 2011). For example, enhanced tumor trafficking was
achieved when anti-GD2 CAR T cells were co-transduced with the chemokine receptor
CCR2b (Craddock et al., 2010). Persistence of CAR-T cells can be achieved by using
gamma-retrovirus and lentivirus that result in stable integration into genome. Persistence
after T-cell infusion can further be fulfilled by strategies including exogenous cytokine
administration, reversal of anti-survival signals, and avoiding inhibitory signals (Fig. 3).
8
Fig 3. Components of successful immunotherapy of CARs (Mato et al., 2015)
Patients blood is collected, T-cells are separated out and lentiviral vectors containing the
CAR construct are used to infect the T-cells. Transfer of genetic material encoding CAR
occurs and CAR positive population is activated and expanded. After 12 to 14 days these
cells are harvested for infusion. Patients are primed for infusion by undergoing
lymphodepletion.
Mechanism of gene therapy with lentivirus vector
Lentivirus is a genus of viruses belonging to the Retroviridae family. They are used to
deliver genetic information into the host cell genome. Hence they are used to carry a
specific CAR sequence, which will be expressed by the effector cells. They have a unique
ability of being able to infect both dividing and non-dividing cells with high efficiency
9
and low immunogenicity. They are considered good gene delivery vectors as they
produce long-term stable transgenes.
Lentiviral vectors used for gene therapy allow delivery of a desired gene into the host
genome but prevent viral replication, and hence are replication defective. For transfection
into the host cell, the lentiviral gene is disrupted into three parts and packaged into three
separate vectors: one has 5’ LTR to drive expression of package genomic DNA/RNA and
two helper plasmids that encode the structural and envelope proteins. After transfection
the intact virus is generated in the supernatant and has the ability to integrate the desired
gene into host cell genome (Fig. 4).
Fig 4. Production of recombinant lentiviral vectors
Gag and Pol are required for the maturation of virion and Vesicular stomatitis virus
(VSV-G) codes for the fusogenic envelope G glycoprotein. Upon transfection, the
packaging cells produce infectious particles containing the sequences from the transfer
plasmid, which can be used to transduce the target T-cells.
10
Other immune cells used in CAR
Natural killer cells: they are a type of cytotoxic lymphocyte that plays a crucial role in
the innate immune system. NK cells act rapidly in response to infectious agents and
pathogens by triggering cytokine release, and also by causing lysis or apoptosis.
NK cells can be derived from different sources such as peripheral blood (PB), bone
marrow (BM), umbilical cord blood (UCB), unstimulated leukapheresis products
(PBSC), human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).
NK cells are defined as CD56+ and CD3- cells and are subdivided into cytotoxic and
immunoregulatory cells. They are of great clinical interest because they contribute anti-
tumor effects without causing graft-versus-host disease (GvHD) (Glienke et al., 2015).
This makes NK cells a potential candidate for utilization in CAR therapy.
Jurkat-NFAT-eGFP T-cells: are an immortalized line of human T lymphocytes. The
cell line is used to study the expression of various chemokine receptors and acute T- cell
leukemia. They do not possess the ability to kill tumor cells but produce interleukin- 2
(IL-2) upon stimulation. This feature is used to test the interaction of the CAR-T cells
with the target tumor cells (Fig. 5).
11
Fig 5. One example of Jurkat cell activation (Stecha et al., 2015)
In this example, Jurkat cells stably expressing the luciferase gene activate the
cytoplasmic signaling pathway upon interaction with the antigen on the tumor target-
cells. The IL-2/NFAT promoter is turned on and its downstream gene (reporter gene) is
expressed.
Lymphoma
Lymphoma is a group of blood cell tumors that develop from the lymphatic cells; with
symptoms that include enlarged lymph nodes, weight loss, fatigue, fever, itching and
drenching sweats that usually occur at night. Lymphoma is most commonly classified
into Hodgkin lymphomas (HL) and non-Hodgkin lymphomas (NHL). WHO has also
included multiple myeloma as a type of lymphoma.
Multiple Myeloma: It occurs due to the production of abnormal plasma cells (a type of
white blood cell) that builds up in the bone marrow and form tumors in many bones of
12
the body. These abnormal plasma cells are called multiple myeloma cells and as their
numbers increase they outnumber the normal plasma cells that make antibodies to help
the body fight infection and disease (Fig. 6a). MM causes weakening of bones by
disrupting the balance between bone resorption and formation. This leads to a viscous
cycle in which MM supports bone resorption and the latter facilitates increased tumor
growth. TRANCE, a member of the TNF family, is produced by osteoblasts in response
to stimulation by MM cells. This facilitates differentiation of osteoclasts progenitors
furthering bone resorption. Osteoprotegerin produced by different cell types helps block
TRANCE in efforts to maintain the bone resorption and formation balance. CD138 is a
cell surface glycoprotein present in large quantities on, and actively shed from MM cells.
It helps block osteoprotegerin, thus facilitating survival and proliferation of MM cells.
IL-6 is another major growth and survival factor for MM cells (Tricot, 2000) Strategies to
curb malignancy involve the use of bisphosphonates and IL-6 inhibitors.
Fig 6a. Production of abnormal plasma cells by the bone marrow leading to MM
(National Cancer Institute, General Information about Plasma Cell Neoplasms, 2015)
13
CD138 (Syndecan-1)
CD138 (syndecan-1) is a transmembrane heparan sulfate cell surface proteoglycan. The
molecule is important in maintaining cell morphologic features and mediates a number of
cellular functions, like cell-matrix interaction, cell-to-cell adhesion, and most
importantly, cell proliferation and differentiation. It is highly sensitive and specific for
plasmacytic differentiation. Some B cell–lineage hematopoietic neoplasms may also
express the molecule but it is limited to the cell population having morphology similar to
plasma cells.
Normal non-hematopoeitic tissues such as epithelial cells express CD138, typically in
squamous and transitional epithelia. Its expression may be lost when these cells undergo
malignant transformation. Endothelial cells in cell culture are said to produce low levels
of this molecule. Malignant melanomas with epithelioid morphologic features also show
CD138 reactivity (Nunez et al., 2012).
Fig 6b. Summary of CD138 reactivity in normal bone and bone related tumors (Nunez et
al., 2012)
14
CD138 expression was seen in 83% osteoid osteoma/osteoblastoma cases and in 31% of
osteosarcoma cases. No expression was seen in chondrosarcomas, giant cell tumors of
bone, or normal/reactive bone.
Limitation of CARs
There are several limitations of CARs but the most adverse effect is the onset of immune
activation post CAR-T cell infusions called Cytokine Release Syndrome (CRS). Second
generation CARs include the addition of co-stimulatory signaling and this translates to
cytokine production and antitumor responses. The symptoms of CRS include anorexia,
cardiac dysfunction, hepatic failure, high fever and malaise. The cytokines elevated
during this process are interferon-gamma, granulocyte macrophage colony-stimulating
factor, IL-10 and IL-6 (Davila et al., 2014). It has been shown that the severity of CRS is
directly correlated to disease burden at the time of infusion. Methods to detect CRS are
currently being investigated. For example, C-reactive protein is made by hepatocytes in
response to IL-6 and is hence used as a laboratory marker for CRS (Davila et al., 2014).
Corticosteroids and IL-6 receptor blockade mAb are used to reverse CRS without
dampening anti-leukemia effects. Tocilizumab is an FDA approved IL-6 receptor blocker
that is used to reverse the side effects caused by CRS. Further investigations are
underway to confirm whether the use of these receptor blockade mechanisms reverse
CRS without affecting CAR-T anti-tumor effects.
Neurological toxicity has been reported post CD19-specific CAR-T infusion with side
effects including confusion, delirium, aphasia and seizures (Davila et al., 2014,
Kochenderfer et al., 2011). The causative pathophysiology has still not been confirmed,
and it is believed that CAR T-cell toxicity on the central nervous system might be
15
possible.
On-target/Off-tumor recognition is another limitation of this therapy wherein the CAR T-
cells target specific antigens shared by both tumor cells as well as normal tissues. The
severity ranged from manageable B-cell aplasia to severe toxicity such as death. Studies
have shown that lowering the CAR T-cell dose and eliminating the preconditioning step
can reverse these effects. Other limitations of CARs include anaphylaxis, insertional
oncogenesis and graft versus host disease (Fig. 7).
Fig 7. Various limitations of CARs (Bonifant et al., 2016)
16
Objectives of the Project
CAR engineered T-cells offer a promising approach for cancer immune therapy and are
widely being investigated by research groups worldwide. Data from several clinical trials
show that CARs have failed due to immunogenicity and short persistence in vivo.
Promising clinical benefits in B cell malignancies using a CD19 specific CAR with co-
stimulatory domain have been published. CD19 specific CARs are being investigated for
their functionality in treating follicular lymphoma, diffuse large B-cell lymphoma,
chronic lymphocytic leukemia and acute lymphoblastic leukemia. However, malignancies
such as Primary effusion lymphoma (PEL) and multiple myeloma (MM) that express
CD138 tumor associated antigens haven’t been investigated with such enthusiasm.
Limited data regarding CAR-T therapy against CD138 malignancies and its toxicity
exists. The objective of this thesis is evaluate the effectiveness of CAR-T cells against
CD138 malignancies by generating an anti-CD138-CAR, expressing them in T-cells and
functionally evaluating its ability to kill multiple myeloma tumor cells.
17
Materials and methods
Generation of lentiviral anti CD138-CAR construct:
The pLENTI-Blast vector was derived from pLenti6v5gw_lacz vector (Invitrogen;
ThermoFisher Scientific) by removal of the LacZ gene. pLenti-MP2 was a gift from
Pantelis Tsoulfas (Addgene plasmid # 36097) and was used to generate the pLENTI-
EF1α lentiviral vector by replacing CMV promoter with human EF1α promoter using
standard molecular biology techniques. psPAX2 was a gift from Didier Trono (Addgene
plasmid # 12260). The pLP/VSVG envelope plasmid and 293FT-cells were obtained
from Invitrogen (ThermoFisher Scientific). The retroviral vector MSCVneo,
MSCVhygro, and MSCVpac and the packaging vector pKAT were obtained from Dr.
Robert Illaria’s laboratory. The CD138-scFv fragment was ligated with a previously
generated CAR-vector designated as pLenti-EF1∆Xho-1-6-1-MYC-hCD8-h4-1BB-
CD3Ζ-T2A-Pac. Both insert and vector DNA were digested using NheI and MluI
enzyme. Ligated products were transformed into E.coli competent cells and colonies
were screened using colony-PCR select CAR-CD138 positive clones. Plasmid prep
(MIDI from 100ml O/N culture) was then followed by our lab protocol for plasmid
purification. The resulting plasmid was pLenti-EF1∆Xho-CD138-MYC-hCD8-h4-1BB-
CD3Ζ-T2A-Pac construct. The control constructs generated and used are pLenti-
EF1∆Xho-Nhe-4C3-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac and pLenti-EF1∆Xho-Nhe-
ID3-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac
The final CAR constructs consisted of human CD8 signal peptide, fused in frame to the
scFv fragments, a Myc epitope tag, the hinge and transmembrane domain of human CD8,
the cytosolic domain of human 41BB (CD137) receptor, the cytosolic domain of human
18
CD3z, a 2A ribosomal skip sequence and a cDNA encoding a puromycin resistance gene.
Virus generation
Recombinant lentiviruses were generated using HEK-293T cells purchased from ATCC.
To produce VSVG-pseudotyped lentiviral supernatant, 293T cells were first cultured
overnight in DMEM medium, then co-transfected with 10µg antiCD138-CAR or the
empty CAR control vector together with 7.5µg of psPAX2 containing Gag, Pol, Rev, and
Tat genes for virus packaging; and 2µg pLP/VSVG for expression of the virus G
glycoprotein, using calcium chloride and 2x HBS transfection reagent. After 24 h, the
medium was changed. 48 h and 72 h post transfection, medium containing lentivirus
supernatant was harvested. A 0.45µm filter unit was used to filter the supernatant and
remove cell debris. The virus was then concentrated 10-fold by ultra-centrifugation at
18,500 rpm at 4°C for 2 hours. T-cell (XVIVO) medium was then used to dissolve the
virus pellet and stored at -80°C for further use.
Viral Transduction
Preparation of T cells: Isolation of primary T-cells from PBMCs of a healthy donor was
done using CD3 microbeads from Miltenyi Biotech. The isolated T cells were cultured in
XVIVO medium from Lonza supplemented with CD3/CD28 soluble antibodies and
purified recombinant human IL2.
Preparation of NK cells: Immortalized NK-92MI cell line purchased from ATCC was
used. It is an interleukin-2 (IL-2) independent Natural Killer Cell line from peripheral
blood mononuclear cells (PBMCs) from a 50-year-old Caucasian male with rapidly
19
progressive non-Hodgkin's lymphoma. NK media supplemented with beta
mercaptoethanol was used to thaw and keep the cells in a healthy state for transduction.
Preparation of Jurkat cells: The Jurkat-NFAT-eGFP cell line is a human T lymphocyte-
based cell line for analysis of NFAT pathway activation. This cell line is maintained using
RPMI (with 10% MEF FBS) media.
Transduction: T-cells (4 million cells/well/2ml) were plated in a 6-well plate and were
either left un-infected or infected with 300µl of concentrated virus supernatant. 8µg/ml
polybrene (PB) was also added. Cells were centrifuged at 2,800 rpm at 32°C for 90 min
and were left undisturbed at 37°C, 5% CO
2
for about 5-6 hours. After incubation, the
medium containing viral supernatant and polybrene was replaced with fresh T-cell
medium and plates were incubated overnight at 37°C. The same transduction protocol
was repeated twice for two more days. T-cells infected with CAR expressing lentiviral
supernatants were transferred to flasks and selected with 400ng/ml of puromycin for
CAR-positive T-cells.
Similar protocol was followed for transduction of NK cells. 0.5 million cells/well/2ml
were plated in a 6 well plate. NK cells infected with CAR expressing lentiviral
supernatants were selected with 1200ng/ml of puromycin for CAR-positive NK cells.
Similar protocol was followed for transduction of Jurkat-NFAT-eGFP cells. 1 million
cells/well/2ml were plated in a 6 well plate. 1ml of concentrated viral supernatant and
30µl of polybrene (with a final concentration of 8µg/ml) was added and centrifuged.
Jurkat-NFAT-eGFP cells infected with CAR expressing lentiviral supernatants were
selected with 250ng/ml of puromycin for CAR-positive Jurkat-NFAT-eGFP cells.
20
In vitro binding assay
The target cell lines were counted (0.2 million cells/1ml of wash buffer) and incubated
with soluble CD138 supernatant. After extensive washes an enzyme linked reporter assay
was conducted to assess the binding affinity of CD138 TAA present on MM target cell
lines to soluble CD138 scFv (antibody).
Flow cytometry of Myc expression
CAR-expressing primary uninfected Jurkat-NFAT-eGFP cells and Jurkat-NFAT-eGFP
cells expressing CD138-CAR were counted and washed with Phosphate-buffered saline
(PBS) containing 1% Fetal bovine serum (FBS). Approximately 0.1 million cells/ml were
stained with Myc-APC antibody and incubated in dark at 4°C for 1 hour followed by
three washes with cold 1ml PBS-FBS wash buffer. The cells were re-suspended in 400µl
of PBS and acquired using FACSverse flow machine by BD biosciences to check Myc
expression in CAR-positive cells.
Flow cytometry of GFP expression
The target cell lines and Jurkat effector cells expressing CD138 CAR were counted,
plated on a 24 well-plate in the ratio 2:1 and incubated at 37°C for 22 hours. The cells
were then collected and re-suspended in 500µl of PBS. GFP expression in CAR-positive
cells was then acquired using FACSverse flow machine by BD biosciences.
21
In vitro cytotoxicity assay
The target cell lines were co-cultured with puromycin selected T-cells expressing the
CD138-CAR in a 10:1 ratio in a 384-well plate. After 4 hours of co-culture at 37°C,
assay for cell death was conducted using an enzyme release assay. Cytotoxicity assay was
also conducted for NK92MI cells expressing CD138-CAR with MM target cell lines in
the ratio 1:1.
ELISA
ELISA plate (384-well) was coated with capture antibody (50µl per well) diluted to a
working concentration in PBS, left overnight at room temperature. Next day, the plate
was washed three times with wash buffer and incubated with blocking buffer (1% BSA in
PBS) for 2 hours at room temperature. Wells were washed three times prior to adding the
cell supernatants to be assayed for interleukin 2 (IL-2). Following 2 hours of incubation
at room temperature, the plates were washed three times and incubated with detection
antibody diluted in reagent diluent for an additional 2 hours. The plates were washed
again and incubated with streptavidin-HRP conjugate in dark for 20 minutes. Finally
substrate solution (1:1 mixture of color reagent A, H
2
O
2
and color reagent B
Tetramethylbenzidine) was added to each well after washing the plate three times with
wash buffer. The plate was incubated for another 20 minutes and absorbance was read at
650nm.
22
Results
Construction of anti-CD138 CAR plasmids
We generated a CAR construct targeting CD138 protein present, and a control CAR
designated as 4C3. The control CAR does not recognize CD138 antigen. The final
constructs were designated as
pLenti-EF1∆Xho-CD138-MYC-hCD8-h4-1BB-CD3Ζ-T2A-Pac,
pLenti-EF1∆Xho-Nhe-4C3-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac and,
pLenti-EF1∆Xho-Nhe-ID3-Mlu-MYC-CD8TM-BBZ-P2A-T2A-Pac
In each case, complementary DNA sequences encoding the corresponding scFv was fused
to a CD8 signal peptide. It was PCR amplified from synthetic gene fragments and cloned
in frame with a human CD8 signal peptide, a Myc epitope tag, the hinge and
transmembrane domain of human CD8, the cytosolic domain of human 41BB (CD137)
receptor, the cytosolic domain of human CD3z, a 2A ribosomal skip sequence and a
cDNA encoding a puromycin resistance gene (Fig. 8a).
23
Fig 8a. Schematic representation of the CD138-CAR construct: pLenti-EF1∆Xho-
CD138-MYC-hCD8-h4-1BB-CD3Ζ-T2A-Pac
The ligated products were transformed into competent cells and plated on carbenicillin
plates. Recombinant clones were screened by colony PCR followed by agarose gel
electrophoresis (Fig. 8b). Plasmid isolation from positive clones was done using standard
MIDI-Prep protocol. DNA sequence was confirmed through automated sequencing.
Fig 8b. Positive clones detected by colony-PCR. Labels 1-8 represent PCR products
using suspected colonies as templates. NC represents negative control (no template) and
24
PC represents positive control using ligation reaction as template. Plasmid isolated from
positive clone 2 was sequence confirmed and used for all experiments.
Binding Assay for detection of CD138 antigen on MM cell lines
Different cell lines namely, L363, U266 and EJM cell lines were tested for binding to
CD138 scFv (antibody). RAJI cells known to express CD19 was used as a positive
control using FMC63 (scFv against CD19). Each cell line was tested for its interaction
with CD138 scFv (antibody) and FMC63 scFv (antibody) (Fig. 9). Media alone was used
to monitor background non-specific binding.
A
B
25
Fig 9. Binding assay to detect the cell line that possess maximum interaction with CD138
scFv (antibody). (A) Binding of L363 cell line with CD138-scFv and FMC63 scFv.
(B) Binding of U266 cell line with CD138-scFv and FMC63 scFv. (C) Binding of EJM
cell line with CD138-scFv and FMC63 scFv. (D) Binding of RAJI cell line withCD138-
scFv and FMC63 scFv.
Among the tested cell lines, L363 showed highest binding affinity to CD138 antibody as
compared to U266 and EJM. RAJI cells express CD19, which binds strongly to FMC63
antibody; this was used as a positive control. Graph shows strong binding of FMC63 with
RAJI cell line as compared to the others. This shows that apart from RAJI, the other cell
D
C
26
lines do not express FMC63. Instead they express CD138 antigen with L363 showing
highest binding affinity.
Detection of Myc expression in CAR infected Jurkat cells
Using the calcium phosphate transfection method, CAR plasmids were transfected into
HEK-293 cells along with the packaging plasmids, pLP/VSVG and psPAX2. The viral
supernatants were collected 24h and 72h post transfection followed by
ultracenrtrifugation at 19,500rpm for 2 hours at 4°C. The Jurkat-NFAT-eGFP cell line
was infected with the lentiviruses encoding the different CAR constructs in the presence
of polybrene.
In order to select CAR-positive Jurkat-NFAT-eGFP cells, puromycin at a dose of
250ng/ml was used. Approximately, 10-15 days later the uninfected Jurkat-NFAT-eGFP
cells were dead and the CAR-infected Jurkat-NFAT-eGFP cells were able to grow in the
media because the CAR construct contained at Pac gene that conferred resistance to
puromycin. The selection was detected by the change in the color of the media from red-
orange to yellow.
In order to confirm the CAR-infected Jurkat-NFAT-eGFP cells were positively selected
we performed flow cytometry. The cells were stained with Myc-APC antibody to confirm
the expression of CAR in Jurkat-NFAT-eGFP cells. Uninfected Jurkat-NFAT-eGFP cells
were used as negative control (Fig. 10).
27
Fig 10. Flow cytometric analysis to confirm CAR expression on cell surface of Jurkat-
NFAT-eGFP cells. Uninfected or Jurkat-NFAT-eGFP cells infected with different
lentiviral CARs were stained with APC-conjugated Myc antibody to detect Myc tag fused
to CARs. (A) Uninfected control Jurkat-NFAT-eGFP cells. (B) Jurkat-NFAT-eGFP cells
transduced with lentiviral CD138 construct after puromycin selection. (C) Overlay of
28
graphs from (A) and (B) showing the shift in the peak. Uninfected Jurkat-NFAT-eGFP
shown by a dashed black line, and CD138 infected Jurkat-NFAT-eGFP shown in red.
This shift confirms the expression of Myc tag fused to CARs as opposed to the control,
which is not infected with the CAR.
Detection of GFP expression in CAR infected Jurkat cells
Jurkat-NFAT-eGFP cells are engineered to turn on the NFAT pathway when activated.
This activation leads to IL-2 production by turning on the IL-2 promoter that causes
transcription of the IL-2 gene. Other pathways such as NF-κB and STAT are also
activated that in turn lead to more cytokine production. The Jurkat-NFAT-eGFP cells are
engineered in such a way that the IL-2 promoter is constructed upstream of the GFP gene,
therefore NFAT pathway when activated also activates GFP expression. Hence Jurkat-
NFAT-eGFP cells expressing CAR cause increased levels of GFP expression when they
interact with the target antigen on the target cell lines. GFP expression depends on
proximal interaction between target cells and CAR-effector cells.
First, we wanted to test whether Jurkat-NFAT-eGFP reporter cells can effectively induce
GFP expression when infected with CARs, upon interaction with appropriate target cells.
In order to do this we infected Jurkat-NFAT-ee cells with a 1-6-1-CAR and co-cultured
with HEL target cells, known to bind to 1-6-1 antibody. After confirming the efficient
induction of Jurkat-NFAT-eGFP expressing the 1-6-1 CARs upon interaction with HEL
cells we wanted to test whether GFP is induced when Jurkat-NFAT-eGFP cells infected
with CD138-CAR are co-cultured with appropriate MM target cells such as L363 (Fig.
11).
29
A
B
30
Fig 11. Flow cytometric analysis of GFP expression in CAR infected Jurkat cells.
(A) 1-6-1-BBZ-BlastR infected Jurkat-NFAT-eGFP cells showed no expression of GFP
(upper panel), but GFP expression was detected when co-cultured with HEL cell line
(lower panel). (B) CD138 infected Jurkat-NFAT-eGFP cells showed no expression of
GFP (upper panel), but GFP expression was detected when co-cultured with target cell
line L363 (lower panel). (C) Overlays of CAR infected Jurkat cells alone and when co-
cultured with their respective targets. CAR infected Jurkat-NFAT-eGFP shown in orange,
and CAR infected Jurkat-NFAT-eGFP co-cultured with respective targets shown in black.
Flow cytometry analysis for GFP expression showed that 1-6-1-CAR infected Jurkat-
NFAT-eGFP cells showed GFP expression when co-cultured with target cells but
expression was absent when they were cultured in media. This confirms that CAR
infected Jurkat-NFAT-eGFP cells upon interaction with the target antigen turn on GFP
expression.
Flow cytometry analysis for GFP expression showed a shift in peak when effector cells
were co-cultured with MM target cell lines as compared to effector cells alone. This
confirms that the CAR infected Jurkat-NFAT-eGFP cells interact with specific target cell
lines and activate GFP expression. It also confirms that the scFv region of the CAR
C
31
infected Jurkat cells interact with CD138 present on the target cells.
Cytotoxicity Assay
We conducted cytotoxicity experiments with two types of cells namely, NK cells and T-
cells. CD138-CAR was infected into NK cells and T-cells; CAR-NK and CAR-T positive
cells were selected with puromycin and expanded.
Cytotoxicity assay of CAR infected NK cells: The effector CD138-CAR NK-cells were
incubated with L363- target cells for 4 hours (effector: target ratio of 1:1). CD138-CAR
NK cells were also tested with RAJI and U266 cell lines. ID3-CAR infected into NK
cells was used as a control CAR. The extent of cell death was assessed by an enzymatic
release assay. Culture medium was used as a blank in cell death assay. These results
show lysis of the target cells by CAR-NK cells (Fig. 12).
A
32
Fig 12. Cell death assay of CAR-NK cells treated with cancer cells. Different CAR NK-
cells co-cultured with CD19 positive RAJI cells, CD138 positive L363 cells and CD138
negative ID3 cells for 4 hours. (A) Cell death of RAJI cells is seen with CD138 and ID3
CAR infected NK cells. (B) Cell death of L363 cells is highest with CD138-CAR
infected NK cells as compared to the control ID3-CAR. (C) Cell death of U266 cells is
seen with CD138 and ID3 CAR infected NK cells.
The ambiguity in the results can be explained by the non-specific nature of killing that
NK cells possess through their own receptors that are not due to the specific CAR
B
C
33
receptor. Also the NK cells were obtained and expanded from an immortalized cell line,
this may affect the extent of cell death, leading to ambiguous results.
To further investigate the CD138-CAR cytotoxicity on MM cell lines, T-cells from
patients were used. This eliminates non-specific killing and the use of immortalized cell
lines.
Cytotoxicity assay of CAR infected T-cells: The effector CD138-CAR T-cells were
incubated with L363 target cells for 4 hours (effector: target ratio of 10:1). CD138-CAR
T-cells were also tested with RAJI and U266 cell lines. 4C3-CAR infected into T-cells
was used as a control CAR. The extent of cell death was assessed by an enzymatic assay.
Culture medium was used as a blank in cell death assay. These results show lysis of the
target cells by CAR-T cells (Fig. 13).
Fig 13. Cell death assay of CAR-T cells treated with MM cancer cells. Different CAR-T
cells co-cultured with L363 and U266 cells for 4 hours. Cell death of L363 cells is
highest with CD138-CAR infected T-cells as compared to the control 4C3-CAR. Cell
34
death of U266 cells is low with CD138-CAR infected T-cells, almost similar to the
control 4C3-CAR. These results are in correlation with the binding assay, which shows
that the MM cell line L363 shows an increased expression of CD138 surface antigen as
compared to the U266 cells. Therefore the extent of cell death caused by the CD138
CAR-T cells is higher than the U266 cell line.
Interleukin-2 Enzyme-linked Immunosorbent Assay (ELISA)
To detect whether increased cytokine secretion could contribute to the cytotoxic effect of
CD19-CAR, we measured the secretion of IL-2 by ELISA. The supernatant from the
CAR Jurkat-NFAT-eGFP-cells was collected after they had been co-cultured with target
cells (CD138
+
L363 cells) for 22 hours at an effector: target cells ratio of 1:2. CAR-
Jurkat-NFAT-eGFP cells are known to produce IL-2 upon binding and interaction with
target cell lines; we specifically examined its secretion by ELISA. We checked IL-2
secretion by Jurkat-CD138-BBZ-Pac alone to control for background interference.
Jurkat-NFAT-eGFP cells expressing 161-CAR co-cultured with its target HEL cells were
used as a positive control to test for IL-2 production.
When L363 cells are co-cultured with Jurkat-NFAT-eGFP cells expressing CD138-CAR,
IL-2 levels showed a slight increase (Fig. 14), which confirmed that Jurkat cells are
activated upon interaction with target cells and responded by secreting IL-2.
35
Fig 14. IL-2 cytokine secretion by Jurkat-NFAT-eGFP expressing CD138-CAR cells
upon activation with L363 cells. (A) The results demonstrated a slight increase in IL-2
expression when CD138-CAR expressing Jurkat-NFAT-eGFP-cells are activated by
interaction with L363. (B) The results demonstrated an increase in IL-2 expression when
1-6-1-CAR expressing Jurkat-NFAT-eGFP-cells are activated by interaction with HEL.
A
B
36
Discussion
Chimeric Antigen Receptor (CAR) immunotherapy has gained popularity in the recent
years in the field of cancer immunotherapy. Clinical trials in which CAR-T cells are
directed against CD19 receptor have shown extremely promising activity against a
number of B cell malignancies. The effective outcome after treatment correlates with the
expansion and long-term persistence of CAR modified T cells. We wanted to focus on a
CAR construct to treat multiple myeloma, the second most common blood cancer that
involves malignancy caused by cancerous plasma cells. Current treatment options for
MM are focused on reducing the signs and symptoms of disease. In order to address the
need of effective therapeutic options we targeted the CD138 antigen expressed on MM
cells. CD138 is highly expressed on abnormal plasma cells (Kumar et al., 2010) as
opposed to CD19, CD27 and CD81 that are expressed on normal plasma cells. Therefore,
in this project, we focused our efforts in trying to generate a CAR targeting CD138
antigen expressed in multiple myeloma. We screened several MM cell lines for surface
expression of CD138 antigen by conducting the binding assay. These results
demonstrated that the target cell line L363 showed highest binding to CD138 scFv and
hence we chose L363 for our studies. These results corroborate with literature showing
similar data for CD138 expression on L363 and U266 cell lines (Zlei et al., 2007). To
monitor T cell activation upon interaction with target cells, we expressed Myc tagged
CD138-CAR in Jurkat-NFAT-eGFP reporter cells. Surface expression of CD138-CAR
was confirmed by staining with a Myc antibody. Jurkat-NFAT-eGFP cells expressing
CAR cause increased levels of GFP expression when they interact with the target antigen
on the target cell lines. GFP expression depends on proximal interaction between target
37
MM cells and CAR- effector cells. FACS was used to determine GFP expression, which
clearly showed an increase in GFP levels when Jurkat-NFAT-eGFP-CD138-CAR cells
were co-cultured with the MM target cell line, L363. We also tested the activity of
CD138-CAR expressing NK cells and T-cells. CD138-CAR expressing NK cells
demonstrated cell death in L363 cell line. NK cells possess their own receptors that may
cause target cell killing that are not due to the specific CAR receptor. Also the NK cells
were obtained and expanded from an immortalized cell line and this may affect the extent
of cell death. Therefore, to confirm target specific killing, we used primary T-cells
expressing CAR-CD138 to test cytotoxicity. ELISA was also conducted to test production
of IL-2 when co-cultured with L363 target cells. The results demonstrated lysis of L363
cell line and production of IL-2, but it is not robust enough. Therefore, further
optimization of the CAR design may be necessary for clinical translation. This may
include, using a different scFv fragment or changing the hinge/spacer region. We are
planning on manipulating the construct by changing the hinge/spacer region and carry out
similar in vitro experiments. Once optimized, in vivo studies will be conducted.
Collectively, our results demonstrate that anti-CD138 CAR-T cells are active and can
exert cytotoxicity against CD138 positive target cells. Besides CD138, different targets
such as CD20, CD28 or CD38 (Kumar et al., 2010) also expressed on abnormal plasma
cells with differing severity of disease can be studied. CARs can be generated for these
targets that can be investigated for their clinical efficacy either alone or in combination
with CD138-CAR presented here to treat MM.
This research can also be extrapolated to CARs engineered against other surface antigens
present on different types of hematological malignancies.
38
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Abstract (if available)
Abstract
Chimeric antigen receptor (CAR) immunotherapy is a breakthrough in cancer therapy because it allows for the in vitro re-engineering of specific T cell receptors against different types of cancer. With CARs, glycolipid and carbohydrate tumor antigens can be made recognizable by T-cells. Tumor cells become “visible” because of direct recognition by T-cell receptor and moreover CAR is a targeting mechanism that functions independent of MHC type recognition. A vast number of CARs have been developed against hematological and non-hematological malignancies, but the success rate is higher in hematological cancers. Among hematological malignancies, CAR T cells are already in clinical trials to treat B-cell malignancies. Multiple myeloma (MM) is the second most common blood cancer involving malignancy caused by cancerous plasma cells. Plasma cell myeloma (PCM) is a type of multiple myeloma where abnormal plasma cells build up in the bone marrow and lead to tumor formation in various bones of the body. These cancerous cells crowd out the normal plasma cells that help fight infections leading to impairment of immune function and kidney damage. The SEER (Surveillance, Epidemiology, and End Results) data for multiple myeloma published by the National Cancer Institute shows the average life expectancy at 4 years. Treatment for multiple myeloma is focused on therapies that could decrease the clonal plasma cell population and thus decrease the signs and symptoms of disease. There is a need for more effective therapeutic options for MM. CD138 is a cell surface protein expressed specifically on MM and plasma cells. An antibody targeted against this antigen can be successfully used to engineer CAR-T cells for MM immunotherapy. My hypothesis is that CD138 targeting CAR-T cells may have potent anti-MM activity.
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Asset Metadata
Creator
Narasappa, Nell Namitha
(author)
Core Title
Generation and characterization of anti-CD138 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
07/08/2016
Defense Date
06/14/2016
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer immunotherapy,CD138 cells for the treatment of hematologic malignancies,chimeric antigen receptor,hematological malignancies,multiple myeloma,OAI-PMH Harvest,syndecan-1
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Chaudhary, Preet M. (
committee chair
), Maxson, Robert E. (
committee member
), Tokes, Zoltan (
committee member
)
Creator Email
narasapp@usc.edu,nellnarasappa@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-265792
Unique identifier
UC11281046
Identifier
etd-NarasappaN-4530.pdf (filename),usctheses-c40-265792 (legacy record id)
Legacy Identifier
etd-NarasappaN-4530.pdf
Dmrecord
265792
Document Type
Thesis
Format
application/pdf (imt)
Rights
Narasappa, Nell Namitha
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
cancer immunotherapy
CD138 cells for the treatment of hematologic malignancies
chimeric antigen receptor
hematological malignancies
multiple myeloma
syndecan-1